中文摘要：
啟動蛋白組織因子（TF）的激活可能是導致纖維蛋白形成的凝血過程中的關鍵步驟。誘導TF激活的刺激在很大程度上是不確定的。在這里，我們發現氧化還原酶蛋白二硫鍵異構酶（PDI）在體內血栓形成過程中直接促進TF依賴性纖維蛋白的產生。在小鼠頸動脈內皮剝脫后，PDI在損傷部位從粘附的血小板和破裂的血管壁細胞中釋放出來。通過活體熒光顯微鏡測定，PDI的抑制降低了不同體內血栓形成小鼠模型中TF引發的纖維蛋白形成。PDI輸注增加，在血小板粘附減少的情況下，PDI抑制減少了損傷部位的纖維蛋白生成，表明PDI可以直接啟動凝血。在體外，人血小板分泌的PDI有助于激活微囊泡（微粒）上的隱性TF。質譜分析表明，TF的部分細胞外半胱氨酸209是組成型谷胱甘肽化的。混合二硫化物的形成有助于將TF保持在低功能狀態。我們提出，減少的PDI通過將混合二硫化物和游離硫醇異構化為分子內二硫化物來激活TF。我們的研究結果表明，二硫化物異構酶可以作為損傷反應信號，觸發血管損傷后纖維蛋白形成的激活。
英文摘要：
The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.
論文信息：
論文題目：
Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation
期刊名稱：J Clin Invest.
時間期卷：2008;118(3):1110-1122.
在線時間：2008年2月14日
DOI：doi.org/10.1172/JCI32376.
產品信息：
貨號：101-A
規格：100ug
品牌：Virogen
產地：美國
名稱：Anti-Glutathione mAb 100
授權現貨代理：Target Technology（靶點科技）
Virogen品牌谷胱甘肽抗體Anti-Glutathione助力蛋白二硫鍵異構酶（PDI）研究，美國Virogen貨號101-A見刊于JCI：
Virogen現貨谷胱甘肽抗體101-A助力蛋白質二硫鍵異構酶研究發表在JCI的材料和方法：
Virogen現貨谷胱甘肽抗體101-A助力蛋白質二硫鍵異構酶研究發表在JCI的材料和方法：
Immunoprecipitation. For the detection of the constitutive glutathionylation of TF, solubilized monocytes were centrifuged 3 times at 3,000 g for 2 minutes at 4°C, and the supernatant was incubated with the monoclonal anti-GSH antibody (Virogen; or control IgG) overnight at 4°C. In other experiments, intact monocytes were incubated with anti-GSH antibody for 60 minutes at room temperature. Then, protein A Sepharose (Sigma-Aldrich; 50 μl) was added and the mixture incubated for 60 minutes at 4°C. Immunoprecipitated complexes were collected by centrifugation at 3,000 g for 2 minutes at 4°C. After washing 3 times with PBS, the pellets were incubated in SDS sample buffer (without reducing reagents) for 60 minutes at 20°C. After centrifugation, the supernatant was subjected to SDS-PAGE (7.5%), followed by electroblotting onto the nitrocellulose membrane. The membranes were then exposed to a monoclonal anti-TF antibody (or control IgG), followed by a horseradish peroxidase–conjugated anti-mouse IgG. The experiments were repeated 3 times.
TF glutathionylation. For the labeling of GSH with biotin, equimolar amounts (10 mM) of NHS-biotin (Pierce Biotechnology) and GSH dissolved in PBS were incubated at room temperature. The reaction was terminated after 1 hour, the residual biotin being quenched with ethanolamine (50 mM). To enable the protein incorporation of the labeled glutathione, the cells (or sTF) were incubated for 30–60 minutes with biotin-GSH (5 mM). Subsequently, the cells were washed and solubilized with 1% Triton X-100 in PBS. sTF was dialyzed using centricon filters. Then streptavidin beads (Sigma) were added to the biotinylated proteins of the cells (or sTF) and the mixture was incubated overnight. After sedimentation of the beads, they were washed twice with PBS containing 0.2% Triton X-100. The proteins were eluted in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, and the monoclonal anti-TF antibody was employed to detect the glutathionylated protein.
